A fundamental problem in neuroendocrinology consists of the locus and/or entity responsible for ultradian rhythms related to the genesis of pulsatile secretory patterns. Recently, we have shown that an immortalized LHRH neuronal cell line (GT1 cells) has the ability to release LHRH in a pulsatile manner, thereby raising the possibility that the LHRH pulse generator may reside within LHRH neurons. The pulsatile secretory activity of these neurons suggests a degree of synchronization of activity to allow for the generation of episodic secretion. Under perfusion conditions, GT1- 7 neurons display a syncytial-like architecture, establishing GAP junctions and synaptic-like contacts. It is still unclear, however, how secretory episodes emerge over an otherwise noisy background of firing cells connected without any apparent predetermined organization. Recent studies have identified nitric oxide(NO) as the endothelium-derived relaxing factor. NO has been localized to neurons and described as an orthodox neurotransmitter based on its particular physico-chemical properties such as an extremely fast diffusibility. We have tested the hypothesis of whether NO is a putative candidate to synchronize LHRH neurons using functional and morphological approaches. Two different strategies were utilized to evaluate the possible participation of NO in the genesis of pulsatile LHRH secretion. Oxyhemoglobin (Hb), a sequester of NO, and NG- monomethyl-L-arginine (L-NMMA), a competitive inhibitor of the NO synthase, were utilized to reduce NO availability or production. GT1-7 cells perfused with medium containing either Hb or L-NMMA showed a dramatic reduction or, in some cases, a complete abolition of LHRH pulsatile activity. Immunocytochemical studies have indicated that these cells stain for citrulline, a collateral product in the biosynthesis of NO by the nitric oxide synthase. These observations support the notion of an effective synthesis of NO in these neurons. In summary, these data indicate that the LHRH neuronal network may utilize NO, at least in part, as a synchronizing mechanism for exhibiting pulsatile LHRH secretion in the absence of any non-LHRH neuronal input.